Benzenesulfonamidoquinolino-β-cyclodextrin as a cell-impermeable fluorescent sensor for Zn2+

Article abstract of DOI:10.1002/asia.200900233

A water-soluble benzenesulfonamidoquinolino-β-cyclodextrin has been successfully synthesized in 30% yield by incorporating a N-(8-quinolyl)- p-aminobenzenesulfonamide (HQAS) group to β-cyclodextrin through a flexible linker. This compound exhibits a good

Full text of DOI:10.1002/asia.200900233

DOI: 10.1002/asia.200900233
Benzenesulfonamidoquinolino-b-cyclodextrin as a Cell-Impermeable
Fluorescent Sensor for Zn2+
Ning Zhang,[a, b] Yong Chen,[a] Miao Yu,[a] and Yu Liu*[a]
Abstract: A water-soluble benzenesul-
fonamidoquinolino-b-cyclodextrin has
been successfully synthesized in 30%
yield by incorporating a N-(8-quinol-
yl)-p-aminobenzenesulfonamide
(HQAS) group to b-cyclodextrin
through a flexible linker. This com-
pound exhibits a good fluorescence re-
sponse in the presence of Zn2+ in
water but gives poor fluorescence re-
sponses with other metal ions common-
ly present in a physiological environ-
ment under similar conditions. Fluores-
cence microscopic and two-dimensional
NMR experiments showed that benze-
nesulfonamidoquinolino-b-cyclodextrin
could bind to the loose bilayer mem-
branes. As a result, benzenesulfonami-
doquinolino-b-cyclodextrin was found
to act as an efficient cell-impermeable
Zn2+ probe, showing a specific fluores-
cent sensing ability to Zn2+-containing
damaged cells whilst exhibiting no re-
sponse in the presence of healthy cells.
Keywords: cell
· cyclodextrins ·
fluorescent probes · NMR spectros-
copy · zinc
Introduction
membrane and have been widely used to detect intracellular
Zn2+ in living cells, and the latter is designed to be excluded
from the contiguous membrane of healthy cells, which have
been successfully used in monitoring cell exocytosis, measur-
ing cell viability, indicating cell apoptosis, and so forth.[12–17]
Among various fluorescent probes, 6-methoxy-(8-p-tolue-
nesulfonamido)quinoline (TSQ) and its derivatives are the
Zinc, following iron, is the second most abundant transition
metal ion present in the body and plays an important role in
various biological processes.[1–2] Zinc deficiency or imbal-
anced zinc distribution within the body, organ, or cell will
lead to a broad range of pathologies, such as neuropathic,
immune, endocrine, and gastro-enterological systems.[3] For
example, the accumulation of intracellular Zn2+ will induce
neuronal injury and lead to neuronal death.[4–5] Therefore,
the detection of Zn2+ in live cells has attracted a lot of at-
tention. It is widely regarded that fluorescent probes, which
allow the visualization of zinc ions in living cells by fluores-
cence microscopy, are one of the best tools for tracking
Zn2+ in cells and organisms.[6–11] Generally, there are two
forms of Zn2+ probes, namely, cell-permeable and cell-im-
permeable probes. The former can pass through the cell
first class of fluorescent probes to be developed for Zn2+
,
which exhibits a high selectivity for Zn2+ as compared to
other metal ions.[18–22] On the other hand, possessing a hy-
drophobic cavity and numerous hydroxyl groups, cyclodex-
trins (CDs), cyclic oligosaccharides with 6–8 d-glucose units
linked by a-1,4-glucose bonds, are widely used as drug carri-
ers and solubilizers. In a preliminary work, we covalently
linked N-(8-quinolyl)-p-aminobenzenesulfonamide (HQAS),
an analogue of TSQ, to b-CD to obtain HQAS-b-CD as a
[23]
water-soluble fluorescent sensor for Zn2+
.
Herein, we
have prepared another HQAS-modified b-CD 1 (Schemes 1
[a] Dr. N. Zhang, Dr. Y. Chen, M. Yu, Prof. Dr. Y. Liu
Department of Chemistry
State Key Laboratory of Elemento-Organic Chemistry
Nankai University, Tianjin, 300071 (P.R. China)
Fax : (+86)22-2350-3625
E-mail: yuliu@nankai.edu.cn
[b] Dr. N. Zhang
Department of Chemistry
Huanghuai University
Zhumadian, 463000 (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.200900233.
Scheme 1. HQAS-modified b-CD.
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hand, the 2-aminoethylamino
group of 1 would be partly
protonated even in a neutral
environment,[27] which is unfav-
orable towards its coordination
with Zn2+
. In addition, the
1H NMR results showed that
the quinoline ring proton (H7)
of 1 exhibits appreciable shifts
(Dd 0.05 ppm), but the other
protons of the substituent of 1
gave no shifts, in the presence
of Zn2+
. Therefore, we de-
Scheme 2. The synthesis method of 1.
duced that the structure of 1/
and 2) as an improved cell-impermeable fluorescent probe
for Zn2+ by the elongation of the spacer between the
HQAS group and the b-CD cavity. In this probe, the HQAS
group of 1 was cell-permeable and could strongly bind Zn2+
.
The b-CD cavity of 1 was hydrophobic and could form a
self-included complex with a quinoline ring, which restricted
the passage of 1 through a healthy cell membrane. In the
case of damaged cells, the junction gap in the cell membrane
became loose, which allowed the b-CD cavity of 1 to include
competitively exposed molecules of the cell membrane and
thus anchor in the loose bilayer of the cell. The long flexible
spacer allows the HQAS group to insert into the hydropho-
bic domains of the loose bilayer of the cell. As a result of
these factors, 1 was expected to act as an efficient cell-im-
permeable Zn2+ probe. This hypothesis was further validat-
ed by fluorescence spectroscopic and fluorescence micro-
scopic investigations, which showed that 1 could selectively
sense the damaged cells but gave no response in the pres-
ence of healthy cells.
Figure 1. Emission spectra (excitation at 360 nm) of 1 ([1]=20 mm) with
the addition of Zn2+ ([Zn2+]=0, 2, 4, 6, 8, 10, 12, 14, 16, 18 mm) in buffer
solution (pH 7.2) at 258C.
Results and Discussion
Fluorescence Properties and Optical Responses to Zn2+
The target product 1 was synthesized in 30% yield through
a typical amide condensation reaction. Arising from the
good solubilization property of the b-CD unit, 1 exhibited
better water solubility and its solubility limit in water could
reach ca. 2 mm. Because the TSQ framework was still re-
tained, 1 exhibited high fluorescence in the presence of
Zn2+. The fluorescence titration curves of 1 with Zn2+
(Figure 1) show that when excited at 360 nm, the emission
intensity of 1 continuously increases upon the stepwise addi-
tion of Zn2+, which demonstrates the possibility of 1 as a
Zn2+ sensor. The coordination stoichiometry of 1 with Zn2+
was investigated by UV/Vis spectrometric titration experi-
ments (see Figure S1 in the Supporting Information), and
Scheme 3. Possible structure of Zn·12.
Zn2+ complex might be similar to that of the HQAS-b-CD/
Zn2+ complex as reported previously.[23] That is, two N
atoms from each of the two HQAS units of 1 participate in
the four-coordinated environment of Zn2+. The binding abil-
ity of 1 to Zn2+ was quantitatively determined by a competi-
tive binding method[28] using the fluorescence titration ex-
periment (see Supporting Information). The apparent stabil-
ity constant (log K, K=[Zn12]/ACTHNUTRGNEUNG
[Zn2+][1]2) of the Zn·12 com-
plex was observed to be equal to 12.6, which was a little
higher than the reported value for the HQAS-b-CD/Zn2+
complex (log K=12.4)[23] but less than that of the Zinquin
acid/Zn2+ complex (log K=13.7).[28] This result unambigu-
the plot of DA1/Zn (DA1/Zn2+ =DA1/Zn ÀA1, where A1 is
defined as the absorption intensity of 1 at 362 nm) vs [Zn2+]/
[1] shows an inflexion point at a molar ratio of 0.5, which
corresponds to a 2:1 coordination stoichiometry between 1
and Zn2+. Therefore, we could deduce the possible structure
of the Zn·12 complex as shown in Scheme 3. On the other
2+
2+
ously demonstrates the strong binding ability of 1 to Zn2+
.
The similar coordination mode may also lead to similar fluo-
rescence enhancement mechanism of 1 as that of HQAS-b-
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Cyclodextrin-based Sensor for Zinc
CD. In this mechanism, two nitrogen atoms of HQAS could
form an intramolecular hydrogen bond with hydrogen atoms
in the absence of Zn2+, which resulted in a photo-induced
electron transfer, and the de-excitation of the resulting tau-
tomer occurs mainly through a non-radiative pathway. As a
result of this process, 1 emits only weak fluorescence. Once
1 coordinated to Zn2+, the electron transfer process was un-
favorable, and an extended p-electron conjugation system,
which involves an internal charge transfer (ICT) process
from ligand donor to Zn2+ acceptor, was formed synchro-
nously.[6,29] Owing to the formation of extended p-electron
conjugation system, the 1/Zn2+ system exhibits an intense
fluorescence.
sured.[30] To study the original conformation of 1 in aqueous
solution, the ROESY experiment was performed at 258C in
D2O. As illustrated in Figure 3, the NOE correlations be-
tween the H3 protons of the quinoline ring and the H5 pro-
The sensing selectivity of 1 to Zn2+ was also investigated
through a comparative study of the fluorescence responses
of 1 to different metal ions. Herein, the effect of other metal
ions was tested by monitoring the emission intensities of 1–
metal systems at 506 nm. The results showed (Figure 2) that
Figure 3. ROESY spectrum of 1 (2 mm) in D2O at 258C.
tons of b-CD (peak A), as well as the NOE correlations be-
tween the H6/H7 protons of quinoline ring and the H3/H5
protons of b-CD (peak B), were clearly observed. These
NOE correlations indicated that the quinoline ring of 1 was
self-included in the b-CD cavity. On the other hand, no
NOE correlations between the protons of the quinoline ring
and the interior protons of b-CD could be found in the two-
dimensional NMR spectrum of the Zn·12 complex, which in-
dicated that the self-included substituent group of 1 had
been excluded from the b-CD cavity after coordination with
Zn2+. Moreover, a molecular modeling study was also per-
formed to support the self-included conformation of 1. The
results (see Supporting Information) showed that the quino-
line ring was located in the interior of the b-CD cavity with
the H3/H6/H7 protons of the quinoline ring located near the
H3/H5 protons of the b-CD cavity, which consequently con-
firmed the estimated self-included conformation of 1.
Interaction of 1 with liposome. It is necessary for cell-im-
permeable probes to possess a suitable affinity for the bio-
logical membrane, in order to prevent the rapid disappear-
ance of fluorescence from stained domains through diffu-
sion. Generally, double-chained phospholipids can assemble
into a bilayer conformation, wherein the hydrophilic polar
head groups orient towards the aqueous environment and
the hydrophobic tail regions orient towards the center
where they are shielded from the aqueous environment.
Spherical lipid bilayers surrounded by an aqueous phase are
called liposomes. Biomembranes are composed mainly of
phospholipid, cholesterol, and protein. So the liposomes of
lecithin and cholesterol have widely been used as biological
membrane models to aid in understanding membrane
Figure 2. Relative fluorescence intensities of 1 ([1]=20 mm) in the pres-
ence of various cations ([Na+]=[K+]=[Ca2+]=[Mg2+]=5 mm, [Zn2+]=
[Cu2+]=[Ni2+]=[Co2+]=[Fe2+]=[Mn2+]=10 mm. These data were mea-
sured at pH 7.2 (lex =360 nm, lem =506 nm)
the addition of a large excess (250 equiv) of alkaline metals
or alkaline earth metal ions (Ca2+, Mg2+, Na+, and K+),
which always exist at a high concentration in living cells,
only slightly change the fluorescence intensity of 1. Similar
phenomena were also observed with the addition of an
equivalent amount of transition metal ions including Fe2+
,
Co2+, Mn2+, and Ni2+. These results jointly demonstrated
the good fluorescent sensing selectivity of 1 to Zn2+. Al-
though Cu2+ quenched the fluorescence of 1, its interference
in the fluorescence response may be masked with a copper
binding protein such as bovine serum albumin.
Conformation of 1. Two-dimensional NMR spectroscopy
has recently become an important method for the investiga-
tion of not only the interaction between mono-modified
CDs and guest molecules, but also the self-included mode
between the CD cavity and its substituting groups. While
guest molecules or substituent groups are included into the
b-CD cavity, the NOE correlations between the protons of
the guest molecules (or substituent groups) and the interior
protons of the b-CD cavity (H3 and H5) will be mea-
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Y. Liu et al.
FULL PAPERS
chemistry. Herein, the interactions of 1 with lecithin lipo-
somes were also investigated by two-dimensional NMR
spectroscopy. As seen from the ROESY spectrum of 1
(2 mm) in a mixture of lecithin, cholesterol, and sodium de-
oxycholate (Figure 4), besides the NOE correlations that
30 min. At the initial stage, only some damaged cells (in-
cluding splinter and dead cells) exhibited weak fluorescence
at cell division domains (Figure 5a and b) but most of the
yeast cells did not exhibit any appreciable fluorescence. In-
Figure 5. a) Optical and b) fluorescence microscopic images of yeast cells
in the presence of 1 (50 mm), c) optical and d) fluorescence microscopic
images of yeast cells in the presence of 1 (50 mm) and ZnSO4 (50 mm),
e) optical and f) fluorescence microscopic images of killed yeast cells in
the presence of 1 (50 mm) and ZnSO4 (50 mm).
terestingly, with time, more and more cells began to emit
green fluorescence. This phenomenon may point to a unique
fluorescent response mechanism of 1 with damaged cells.
Possessing a compact and integrated cell membrane, the
healthy cells had the ability to exclude cell-impenetrable
molecule.[15] The self-included conformation of 1 also re-
stricted the interaction of 1 with cell membranes. Therefore,
1 gave no response with healthy cells. However, with dam-
aged cells, the junction gap in the cell membrane increased,
which allowed the b-CD cavity of 1 to include competitively
exposed molecules of the cell membrane and anchor in the
loose bilayer of the cell. Subsequently, the free HQAS
group could settle in the cell membrane and combine with
the intracellular Zn2+ to give green fluorescence. On the
other hand, when the cells were transferred from the incu-
bated solution to the sample solution for a long time, the
cells gradually died. Therefore, the damaged yeast cells that
had lost their membrane integrity would allow 1 to fluoresce
by catching Zn2+. Interestingly, when we increased Zn2+
concentration by adding extra Zn2+ solution to the 1-treated
yeast cells, the damaged cells exhibited bright green fluores-
cence owing to the significant fluorescence enhancement of
the 1–Zn2+ system as described above, but the healthy cells
still gave no fluorescence signal (Figure 5c and 5d). In an-
other experiment, the incubated solution of yeast cells was
heated to boil for 30 min to kill the cells. Then these dead
cells were separated from the solution by centrifugation and
transferred to the solution containing Zn2+ and 1. The fluo-
rescence microscopic images were recorded under the same
conditions. The results showed that all of the cells (contain-
ing the budding cells and the candida cells) presented strong
fluorescence (Figure 5e and 5 f). These observations jointly
Figure 4. ROESY spectrum of 1 (2 mm) in the presence of liposome at
258C.
originate from the self-inclusion of 1 (peaks C), the protons
whose d value ranged from 0 to 2 (including cholesterol pro-
tons, sodium deoxycholate protons, and the methylene pro-
tons in the alkyl chain of lecithin) presented strong and
complicated NOE correlations (peaks D) with the H3/H5
protons of b-CD. Our previous studies demonstrated that
the H3/H5 protons of b-CD exhibited NOE correlations
with the protons of sodium deoxycholate in a micelle
system,[31] but their intensity was far weaker than that shown
in Figure 4. Therefore, we deduced that the NOE correla-
tions between cholesterol protons or lecithin protons in lipo-
some and the H3/H5 protons of b-CD would also exist.
Since the destroyed liposome had a structure similar to the
damaged cell membranes, these results indicated that the b-
CD cavities of 1 had the capability of including cholesterol
or lecithin of the damaged cell membranes.
Fluorescence microscopy. Yeast cells have many features
analogous to mammalian cells. When stained with Zinquin,
the intracellular labile zinc pool of yeast cells appeared to
localize to small punctuated cytoplasm vesicles, similar to
the zincosomes described in mammalian cells.[32] Therefore,
yeast cells were used as the substrate to investigate the
Zn2+-response ability of 1 in cells. In our experiments, the
pre-cultured yeast cells were transferred to a solution of 1,
and fluorescence microscopic images were recorded after ca.
1700
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Cyclodextrin-based Sensor for Zinc
validated the specific fluorescent sensing of 1 to the Zn2+
containing damaged cells. It was noteworthy that, because 1
could not adhere to a smooth and integrated surface, the
glucose crystals in Figure 5e could not be stained.
-
was collected by filtration, washed with ethanol, and subsequently puri-
fied twice on a Sephadex C-25 column with 1m ammonia as eluent. After
drying in vacuo, a pale yellow product (460 mg) was obtained in 30%
yield. 1H NMR (D2O, 300 MHz, TMS): d=2.25-2.92 (m, 6H), 2.94-4.12
(m, 44H), 4.92 (s, 7H), 7.25 (m, 2H), 7.39 (s, 1H), 7.55 (d, J=7.8 Hz,
2H), 7.66 (s, 1H), 7.79 (d, J=7.8 Hz, 2H), 8.27 (s, 1H), 8.80 ppm (s,
1H); MS (ESI): m/z (%) calcd: 1558.51 [M+H]+; found: 1558.63; ele-
mental analysis: calcd (%) for C63H91N5O38S·6H2O: C 47.69, H 6.54,
N 4.41; found: C 48.05, H 6.45, N 4.69.
This supposition was also proved by the experiments with
stained splinter cells, which showed that 1 stained only the
division domains of the splinter cells but not other regions
(see Supporting Information). A possible reason for this ob-
servation may be that the junction gap in the splinter cell
membrane was loose, which allowed 1 to include cholester-
ols of the cell membrane. Moreover, the HQAS group of 1
was also inclined to insert into the hydrophobic regions of
the bilayers.[33] These reasons jointly enabled 1 to anchor at
the loose bilayer of the damaged cell membrane. In the case
of the compact and integrated cell membrane of living cells,
1 could not include any molecules or anchor on the cell sur-
face.
Yeast Cell Culture and Cell Staining
Yeast (saccharomyces cerevisiae) was obtained from the Agronomy &
Forestry Department of Huanghuai University and used as a cell model.
Prior to use, it was dispersed on a YPD plate (1.0% yeast extract, 4%
peptone, 2.0% glucose, 0.2% (NH4)2SO4) and cultured for 2 d at 358C.
For yeast staining, some colonies were selected and added to a solution
of 1 (50 mm). Subsequently, the cells were washed twice with distilled
water to remove uncombined 1 by centrifugation. A suitable volume of
pre-cultured cells was diluted with or without the addition of ZnSO4
(50 mm). Cells for experiments were dropped on glass slides and covered
with a cover glass. All imaging experiments were performed on an YZ-2
fluorescence microscope (Beijing Keyi Electro-optic Plant) equipped
with a 100W/DC mercury lamp for UV excitation and a SPC-382B color
CCD camera for photo collection. The total magnification was 400ꢁ.
Conclusion
Liposome Preparation
In conclusion, we successfully prepared a water soluble and
cell-impenetrable zinc probe, which could selectively sense
the Zn2+-containing damaged cells. This property will
enable its potential application in the measurement of cell
Lecithin liposome was prepared by a method similar to that described by
Wertz et al.[26] The mixture of egg phosphatidylcholine (60 wt.%), choles-
terol (20%), and sodium deoxycholate (20%), which increased the solu-
bility of lecithin and keep the solution clear, were dissolved in chloro-
form/methanol (2:1). The mixture was placed in a culture tube and the
solvent was removed with a stream of nitrogen and then dried under
high vacuum at room temperature. The solid matter was dissolved in
D2O to provide a final lecithin concentration of ca. 2 gdmÀ3. The suspen-
sions were sonicated at 608C for ca. 15 min until the solution became
clear.
viability or detection of cell apoptosis involving Zn2+
.
Materials and Methods
General
All chemicals were of reagent grade and used as received unless other-
wise specified. N-(8-quinolyl)-p-aminobenzenesulfonamide (HQAS)[24]
and mono[6-(2-aminoeythylamino)-6-deoxy]-b-CD[25] were prepared ac-
cording to reported methods. Fluorescence spectra were measured in a
Acknowledgements
We thank 973 Program (2006CB932900), NNSFC (Nos. 20721062 and
20772062), Tianjin Natural Science Foundation (07QTPTJC29600), and
Key Project of Chinese Ministry of Education (No 107026) for financial
support.
conventional rectangular quartz cell (10ꢁ10ꢁ45 mm) at 258C on
a
JASCO FP750 spectrometer equipped with a constant-temperature water
bath. Tris-HCl buffer solution (pH 7.2) was used as the solvent in all
spectral measurements. NMR experiments were recorded on a Bruker
300 instrument.
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HQAS-succinic acid: To succinic anhydride (3.0 g, 30 mmol) dissolved in
50 mL of dry toluene, was added HQAS (3.0 g, 10 mmol). The mixture
was stirred at 1508C for 5 h. After cooling to room temperature, the pre-
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1H NMR ([D6]DMSO, 300 MHz, TMS): d=12.13 (s, 1H, -COOH), 10.29
(s, 1H, Quinoline-NH-), 9.79 (s, 1H, Phene-NH-), 8.86 (m, 1H, H4 of
Quinoline), 8.33 (d, J=8.1 Hz, 1H, H2 of Quinoline), 7.25–7.95 (m, 8H,
Quinoline and Phene), 2.50 ppm (s, 4H, -CH2-CH2- overlapped with the
peaks of DMSO); MS (ESI): m/z (%) calcd: 398.09 [MÀH]À, 797.18
[2MÀH]À; found: 398.50, 797.30; elemental analysis: calcd (%) for
C19H17N3O5S: C 57.13, H 4.29, N 10.52; found: C 56.95, H 4.07, N 10.65.
1: To
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